Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones: Synthesis, biological activity and SARs

Abstract

Earlier studies had confirmed that the 7-phenylpyrroloquinolinone (7-PPyQ) nucleus was an important scaffold for new chemotherapeutic drugs targeting microtubules. For wide-ranging SARs, a series of derivatives were synthesized through a robust procedure. For comparison with the reference 3-ethyl-7-PPyQ 31, the angular geometry and substituents at the 3 and 7 positions were varied to explore interactions inside the colchicine site of tubulin. Of the new compounds synthesized, potent cytotoxicity (low and sub-nanomolar GI₅₀ values) was observed with 21 and 24, both more potent than 31, in both leukemic and solid tumor cell lines. Neither compound 21 nor 24 induced significant cell death in normal human lymphocytes, suggesting that the compounds may be selectively active against cancer cells. In particular, 24 was a potent inducer of apoptosis in the A549 and HeLa cell lines. With both compounds, induction of apoptosis was associated with dissipation of the mitochondrial transmembrane potential and production of reactive oxygen species, indicating that cells treated with the compounds followed the intrinsic pathway of apoptosis. Moreover, immunoblot analysis revealed that compound 24 even at 50 nM reduced the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1. Finally, molecular docking studies of the newly synthesized compounds demonstrate that active pyrroloquinolinone derivatives strongly bind in the colchicine site of β-tubulin.

1. Introduction

A major difference between cancer cells and many healthy cells is that transformed cells often divide much faster. The microtubule (MT) cytoskeleton is highly dynamic in mitosis, and therefore dividing cells are particularly susceptible to agents that target tubulin [1]. Colchicine site inhibitors exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation [2]. Despite significant structural diversity, colchicine site inhibitors are believed to act by a common mechanism via binding to the colchicine site on tubulin. Among them, we can include 7-phenyl-pyrroloquinolinones (7-PPyQs) and, especially, the 3-substituted derivatives bind to the site with high affinity, inhibiting microtubule assembly and therefore producing a strong antiproliferative effect. Their activities are similar to those of the reference compound combretastatin A-4 [3–9]. The more cytotoxic 7-PPyQ derivatives showed remarkably in vitro biological properties [5] and good antitumor activity in vivo [3]. Some 7-PPyQs, characterized by alkyl substituents at the pyrrole nitrogen, show increased cytotoxicity with nanomolar and sub-nanomolar GI₅₀ values and overcome the resistance observed with the clinically used drugs vincristine and taxol [4]. In an effort to produce additional highly active compounds, numerous related analogs were designed, synthesized and studied, resulting in the discovery of potent 3N-acyl derivatives showing low nanomolar GI₅₀ values and an anti-tubulin mechanism profile similar to that previously observed [8]. Moreover, the most recent 3-benzoyl-7-PPyQ is even more cytotoxic than the initially reported compounds. Moreover, the 3-benzoyl-7-PPyQ has minimal toxicity for nontumor cell lines, good in vitro metabolic stability and synergizes with conventional chemotherapeutic agents in inhibiting leukemia cell proliferation [9]. Through a tubulin docking study with 7-PPyQ amide derivatives at the colchicine site, we observed a hydrogen bond involving the 6-NH and strong hydrophobic interactions due to the pyrrolo[3,2-f]quinolinone scaffold and to the phenyl ring at position 7. The substituents at the 3N-pyrrole were placed in a pocket formed by numerous amino acid residues. This binding mode is compatible with a competitive mechanism of action at the colchicine site.

In the present study, we enrich the available library of pyrroloquinolinone derivatives to obtain greater knowledge of SARs for further development of PPyQs and to understand better how PPyQs are accommodated in the colchicine site pocket.

The reference compound, 3-ethyl-7-phenyl-3H-pyrrolo[3-2-f] quinolin-9-one (31, Fig. 1) was our start point for chemical modifications [4]. Compound 31 has the elements determined to be crucial for anti-tubulin activity, such as the [3,2-f] configuration, the 7-phenyl and 9-carbonyl groups and the ethyl group at position 3. All these elements were important for good antiproliferative effects. This study explores the role of larger 3 and 7 substituents and various scaffold geometries. Among our most interesting findings were that placement of a naphthalene moiety at the 7 position, a 3N-n-octadecyl alkyl chain and [3,2-g] and [3,2-h] geometries led to enhanced cytotoxicity. The synthesis and biological activities of the new compounds are summarized in this report.

Fig. 1.

Structure-activity relationships of 7-pyrrolo[3,2-f]quinolinones. The three main structural determinants for potent cytotoxicity modified for obtaining novel PyQs were the 3- and 7-substituents and the angular geometry.

2. Results and discussion

2.1. Chemistry

The 4-step method leading to 7-PPyQ compounds was previously described [4] (Scheme 1). First, commercially available 5-, 6- or 7-nitroindoles were subjected to an N-alkylation reaction using appropriate halogenated compounds to obtain the nitroindole derivatives 1–4. The same reaction conditions were used with the chloro- and bromo-compounds in anhydrous DMF in the presence of NaH, at room temperature with the ethyl bromide and 50 °C with the n-octadecyl chloride, resulting in high yields of the reaction products. The catalytic reduction (Pd/C 10%, H₂ at atmospheric pressure, ethyl acetate) of intermediates 1–4 gave the corresponding aminoindoles 5–8 in almost quantitative yields. The non-commercially available β-ketoesters 9–12 were prepared following a method reported in the literature [10] by reacting the appropriate methyl-aryl ketone and diethyl carbonate in the presence of NaH, in anhydrous dioxane at 80 °C for 6 h, with good yields. Next, in order to obtain the enamine derivatives 13–18 as condensation products of the reactions between the β-ketoesters and aminoindoles, compounds 5 and 6 were reacted with commercial ethyl acetoacetate, benzoyl acetate and the prepared compounds 9–12. All the reactions were carried out in absolute ethanol at reflux in the presence of a catalytic amount of acetic acid, yielding the acrylate derivatives as crude material 13–18. These had to be purified by silica gel column chromatography before being submitted to thermal cyclization in boiling diphenyl ether (250 °C) to obtain the final products 19–24. Using the same reaction conditions, enamines 25 and 28 were also obtained by reacting 3-ethyl-6-aminoindole (7) and 3-ethyl-7-amino-indole (8) with benzoyl acetate. These compounds were also purified before being cyclized to the tricyclic products. In the case of enamine 25, an un-resolvable mixture of two cyclized compounds was obtained. By NMR spectroscopy it was possible to identify compound 26 as a [2,3-f] angular and 27 as a [3,2-g] linear PPyQ. In the same way, from enamine 28 a mixture was obtained that was separated giving the two compounds in the scheme: the angular [3,2-h] PPyQ 29 and the [1,4]diazepin-indole derivative 30. All cyclization products were purified by re-crystallization from a suitable solvent or by flash chromatography, with their purity verified by HPLC (>95%).

Scheme 1.

a) NaH, NaI, bromoethane or n-octadecyl-Cl, DMF, 50 °C, 6 h; b) H2, Pd/C 10%, EtOAc, 55 °C, 12 h; c) NaH 60%, anhydrous dioxane, 80 °C, 6 h; d) abs EtOH, AcOH, drierite, 100 °C, 48 h; e) Ph2O, 250 °C, 15 min.

2.2. Biological evaluation

2.2.1. In vitro antiproliferative activities and SAR analysis

The new compounds were designed to obtain additional SAR information by modifying the size of substituents at the 3 and 7 position of the aryl-PyQ tricycle, as well as the angular geometry. Evaluation of antiproliferative activities of 19–24, 26–27, 29 and 30 was performed with the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay against a panel of 7 human tumor cell lines (HeLa, A549, HT-29, MDA-MB-231, RS4; 11, Jurkat, and Kasumi-1). GI₅₀ values, the concentrations that inhibit cell growth by 50%, are presented in Table 1. As reference compound CA-4 was also added. From the cytotoxicity data reported in the Table, we can make some relevant remarks on SARs on the changed structural elements with respect to reference compound 31 (Fig. 1). Regarding the 3-substituent on compounds 19 and 20, replacing the ethyl group of 31 with a bulky linear alkyl chain (18 C) caused a massive loss of antiproliferative activity in all cell lines examined. This behavior was expected for 19, which lacks the crucial phenyl in the 7 position, but this loss of activity was unexpected for 20, which has the 7-phenyl group. In past studies [4], very high cytotoxicity was found for compounds with side chains up to 5 C at position 3 with a 7-phenyl. Compounds 21–24, all bearing a 3-ethyl chain as in compound 31, had nanomolar GI₅₀s, with compounds 21 and 24 all being especially active. These compounds all have even bulkier aryl 7-substituents than the 7-phenyl group, with the active 21 and 24 having, respectively, an α-naphthyl and a benzodioxole substituent. In general, for angular [3,2-f] PPyQs such bulky groups are well tolerated. Of particular note, 21 and 24 had greater activity than 31, in both the leukemic and solid tumor cell lines. In contrast, compound 22 with a 7-β-naphthyl substituent was 27-1430-fold less cytotoxic than compound 21, depending on the cell line. In part, this must indicate a less favorable fit into the colchicine site relative to 21. Cytotoxicity again increased with compound 23, which had a 7-tetrahydronaphthyl substituent. Concerning the third structural element taken into consideration in this study, the configuration of the tricycle scaffold, the GI₅₀s obtained with compounds 26, 27 and 29 clearly indicate that the geometries [f] and [g] of 26 and 27, respectively, produce at least one highly cytotoxic compound (nanomolar GI₅₀s), while the [h] geometry does not (compound 29). Compound 30, a diazepine-indole derivative, was also tested, and it was completely inactive (GI₅₀s > 10000 nM).

Table 1.

In vitro cell growth inhibitory effects of compounds 19–24, 26–27 and 29–31.

compound	GI50 (nM)a
	HeLa	A549	HT-29	MDA-MB-231	RS4; 11	Jurkat	Kasumi-1
19	2380 ± 400	3400 ± 400	>10000	>10000	>10000	>10000	>10000
20	8350 ± 440	820 ± 140	>10000	5362 ± 2043	>10000	>10000	>10000
21	0.5 ± 0.03	4.2 ± 0.7	1.9 ± 0.7	17.1 ± 9.4	0.5 ± 0.05	0.7 ± 0.002	0.4 ± 0.1
22	714 ± 2.9	744 ± 26	376 ± 58	465 ± 43	22 ± 2	32 ± 4	396 ± 39
23	34 ± 0.35	45 ± 0.94	4.7 ± 0.5	591 ± 35	7 ± 0.8	3 ± 0.02	15 ± 2
24	4.2 ± 0.3	1.1 ± 0.3	0.6 ± 0.04	123 ± 8.8	0.2 ± 0.05	0.1 ± 0.002	0.3 ± 0.09
26–27	49 ± 0.16	7.1 ± 0.75	16.0 ± 3.3	641 ± 57	13 ± 1.5	27 ± 2	30 ± 2
29	5920 ± 186	9590 ± 9.9	>10000	>10000	>10000	>10000	>10000
30	>10000	>10000	>10000	>10000	>10000	>10000	>10000
31b	11 ± 8	32 ± 1.5	32 ± 1.2	n.d.	2 ± 0.3	0.5 ± 0.2	n.d
CA-4c	4.0 ± 1.0	180 ± 50	3100 ± 100	n.d.	0.8 ± 0.2	5.0 ± 0.6	n.d.

^aIC₅₀ = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SE from the dose-response curves of at least three independent experiments. n. d. not determined.

^bData taken from Ref. 4 and 8.

^cData taken from Ref. 8 and 20.

2.2.2. Evaluation of cytotoxicity of compounds 21 and 24 in human non-cancer cells

To obtain a preliminary indication of the cytotoxic potential of these derivatives in normal human cells, two of the most active compounds (21 and 24) were evaluated in vitro against peripheral blood lymphocytes (PBL) from healthy donors (Table 2). Compound 21 was completely inactive in quiescent lymphocytes (GI₅₀ > 100 µM), while in the presence of the mitogenic stimulus phytohematoaglutinin (PHA), the GI₅₀ was about 25.7 µM. Notably, this value was almost 10000–30000 times higher than that observed against the lymphoblastic cell lines Jurkat and CEM (Tables 1 and 3). These results indicate that 21 has a modest inhibitory effect in rapidly proliferating non-cancer cells but not in quiescent cells, as previously observed for other antimitotic derivatives developed by our group [8,9]. Analogous behavior was observed for compound 24, which showed a GI₅₀ of 45.3 µM in quiescent lymphocytes, while, in the presence of PHA, the GI₅₀ decreased to about 23.0 µM.

Table 2.

Cytotoxicity of compounds 21 and 24 for human peripheral blood lymphocytes (PBL).

	GI50 (µM)a
	21	24
PBLrestingb	>100	45.3 ± 1.4
PBLPHA c	25.7 ± 1.2	23.0 ± 1.5

Values are the mean ± SEM from three separate experiments.

^aCompound concentration required to reduce cell growth by 50%.

^bPBL_resting not stimulated with PHA.

^cPBL_PHA stimulated with PHA.

Table 3.

Cytotoxicity of 21 and 24 in multidrug resistant cells.

	IC50 (nM)a
	CEMwt	CEMVbl100
21	29.8 ± 1.4	2.3 ± 0.4
24	27.8 ± 0.81	3.7 ± 0.9
Vinblastine	1.5 ± 0.3	285 ± 69

Values are the mean ± SEM for three separate experiments.

^aCompound concentration required to reduce cell growth inhibition by 50%.

2.2.3. Effect of compounds 21 and 24 on multidrug resistant cells

To investigate whether these derivatives are substrates of drug efflux pumps, two of the most active compounds (21 and 24) were tested against CEM^Vbl–100 cells that are a multidrug-resistant line selected against vinblastine [4] and that overexpress P-glycoprotein (P-gp). This membrane protein acts as a drug efflux pump and exhibits resistance to a wide variety of structurally unrelated anticancer drugs and other compounds. As shown in Table 3, both compounds exhibited cytotoxic activity in the CEM^Vbl-100 cell line that was even higher than their activity against the parental line. Thus, these derivatives are not substrates for P-gp.

2.2.4. Inhibition of tubulin polymerization and colchicine binding

To evaluate the tubulin interaction properties of compounds 21, 23, 24 and of the mixture 26–27, we investigated their effects on the inhibition of tubulin polymerization and on the binding of [³H] colchicine to tubulin (Table 4) [11–13]. For comparison, CA-4 was examined in contemporaneous experiments as a reference compound along with compound 31. Among the test compounds, 21 and 24 strongly inhibited tubulin assembly with IC₅₀ below 1 µM (0.99 and 0.84 µM, respectively) but slightly higher than that obtained for the reference compounds CA-4 (IC₅₀ = 0.64 µM) and 31 (IC₅₀ = 0.57 µM). Compound 23 also inhibited tubulin assembly with a low IC₅₀ value of 1.1 µM, while the mixture 26–27 had less activity, with an IC₅₀ of 6.2 µM. The PPyQ compounds all inhibited the binding of [³H]colchicine to tubulin, with the best activity occurring with 21 and 24, but none approached CA-4 in its potency as an inhibitor of colchicine binding. These results with tubulin correlate well with the growth inhibitory effects exhibited by compounds 21, 23 and 24, indicating that their antiproliferative activity derives mostly from an interaction with tubulin. For the mixture 26–27, the correlation between cytotoxicity and inhibitory effects on tubulin is perhaps not as good and could indicate there is an additional mechanism of action for this compound.

Table 4.

Inhibition of tubulin polymerization and colchicine binding by compounds 21, 23, 24 and 26–27.

Compound	Inhibition of tubulin assembly IC50 (µM) ± SDa	Inhibition of colchicine binding % Inhibition ± SDb
21	0.99 ± 0.07	46 ± 2
23	1.1 ± 0.1	26 ± 0.6
24	0.84 ± 0.05	49 ± 2
26–27	6.2 ± 0.5	24 ± 4
31c	0.57 ± 0.02	73 ± 0.7
CA-4	0.64 ± 0.01	98 ± 0.1

^aInhibition of tubulin polymerization. Tubulin was at 10 µM.

^bInhibition of [³H]colchicine binding. Tubulin and colchicine were at 1 and 5 µM, respectively.

^cData taken from Ref. 4.

2.2.5. Influence of test compounds 21, 23 and 24 on the cell cycle

The effect of compounds 21, 23 and 24 on cell cycle progression was examined by flow cytometry in Hela cells (Fig. 2). After a 24 h treatment, all compounds induced a G2/M arrest, although compound 21 showed a more modest accumulation in G2/M cells than occurred with the other compounds. In contrast, compounds 23 and 24 induced a greater G2/M block even at lower concentrations. A concomitant reduction of both the S and G1 phases was also observed.

Fig. 2.

Percentage of cells in each phase of the cell cycle in HeLa cells treated with compounds 21 (A), 23 (B) and 24 (C) at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the Experimental Section. Data are presented as mean of two independent experiments ± SEM.

We also studied the association between 24-induced G2/M arrest and alterations in G2/M regulatory protein expression in HeLa cells. As shown in Fig. 3, compound 24 caused, in a time- and concentration-dependent manner, an increase in cyclin B1 expression after 24 and 48 h, indicating an activation of the mitotic checkpoint following drug exposure. This effect was confirmed by a reduction in the expression of phosphatase cdc25c at 24 h, followed by a disappearance in its expression at 48 h. This was associated with the appearance of slower migrating forms of phosphatase cdc25c indicative of cdc25c phosphorylation. The phosphorylation of cdc25c directly stimulates its phosphatase activity, and this is necessary to activate cdc2/cyclin B on entry into mitosis [14,15]. We also observed a decrease of the phosphorylated form of cdc2 kinase, in particular after the 48 h treatment.

Fig. 3.

Effect of compound 24 on cell cycle checkpoint proteins. HeLa cells were treated for 24 or 48 h with the indicated concentrations of 24. The cells were harvested and lysed for detection of the expression of the indicated protein by western blot analysis. To confirm equal protein loading, each membrane was stripped and reprobed with anti-γ-tubulin antibody.

2.2.6. Compounds 21, 23 and 24 induce apoptosis in different cell lines

To evaluate the mode of cell death induced by test compounds, we performed a bi-parametric cytofluorimetric analysis using propidium iodide (PI) and annexin-V-FITC, which stain DNA and phosphatidylserine (PS) residues, respectively. We used two cell lines, Hela and A549, in which we evaluated the effects of compounds 21, 23 and 24. In additional experiments we have also evaluated the apoptotic response of CA-4, as reference compounds, in HeLa cells. As shown in Fig. 4, the three compounds had different potencies. In both cell lines, the compounds induced apoptosis in a time and concentration dependent manner, with compounds 21 and 24 being the most active compounds in good agreement with the cytotoxicity data. To note, that CA-4 induce a strong apoptotic response at the concentration of 0.1 µM in HeLa cells in well agreement with previous reports [16,17].

Fig. 4.

Flow cytometric analysis of apoptotic cells after treatment of A549 (Panels A and B) and HeLa cells (Panel C and D) with 21, 23 or 24 at the indicated concentrations after incubation for 24 (A,C) or 48 h (B,D). The induction of apoptosis was also evaluated in Hela cells (panel E) with CA-4 (0.1 µM) chosen as reference compound. The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are presented as mean ± SEM of three independent experiments.

2.2.7. Compound 24 induced mitochondrial depolarization and reactive oxygen species (ROS) production

Mitochondria play an essential role in the propagation of apoptosis [18,19]. It is well established that, at an early stage, apoptotic stimuli alter the mitochondrial transmembrane potential (Δψ_mt). Δψ_mt was monitored by the fluorescence of the dye JC-1 [20]. HeLa cells treated with compound 24 (0.1–1.0 µM) showed a time-dependent increase in the percentage of cells with low Δψ_mt (Fig. 4, Panel A). The depolarization of the mitochondrial membrane is associated with the appearance of annexin-V positivity in the treated cells when they are in an early apoptotic stage. In fact, the dissipation of Δψ_mt is characteristic of apoptosis and has been observed with both microtubule stabilizing and destabilizing agents, including other derivatives, in different cell types [21–24]. It is well known that mitochondrial membrane depolarization is associated with the mitochondrial production of ROS [25,26]. Therefore, we investigated whether ROS production increased after treatment with the test compounds. We utilized the fluorescence indicator 2,7-dichlorodihydrofluorescein diacetate (H₂-DCFDA) [27]. As shown in Fig. 4 (Panel B) compound 24 induced significant production of ROS starting at 12–24 h of treatment at 1 µM, in good agreement with the mitochondrial depolarization described above.

2.2.8. Compound 24 induced PARP activation and caused a decrease in the expression of anti-apoptotic proteins

As shown in Fig. 5, compound 24 in HeLa cells caused a concentration and time-dependent cleavage of poly (ADP-Ribose) polymerase (PARP), confirming its pro-apoptotic activity.

Fig. 5.

Panel A. Assessment of mitochondrial membrane potential (Δψ_mt) after treatment of HeLa cells with compound 24. Cells were treated with the indicated concentration of compound for 6, 12, 24 or 48 h and then stained with the fluorescent probe JC-1 for analysis of mitochondrial potential. Cells were then analyzed by flow cytometry as described in the Experimental Section. Data are presented as mean ± SEM of three independent experiments. Panel B. Assessment of ROS production after treatment of HeLa cells with compound 24. Cells were treated with the indicated concentration of compound for 6, 12, 24 or 48 h and then stained with H₂-DCFDA for the evaluation of ROS levels. Cells were then analyzed by flow cytometry as described in the Experimental Section. Data are presented as mean ± SEM of three independent experiments.

We also investigated the expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1. Bcl-2 plays a major role in controlling apoptosis through the regulation of mitochondrial processes and the release of mitochondrial proapoptotic molecules that are important for the cell death pathway [28–30]. Our results (Fig. 6) showed that the expression of the anti-apoptotic protein Bcl-2 was decreased starting after a 24 h treatment at both concentration used (0.1 and 1.0 µM). The decrease in expression of Mcl-1 was even greater.

Fig. 6.

Western blot analysis of Bcl-2, Mcl-1 and PARP after treatment of HeLa cells with 24 at the indicated concentrations and for the indicated times. To confirm equal protein loading, each membrane was stripped and reprobed with anti-γ-tubulin antibody.

2.3. Computational studies

Molecular docking studies were carried out to investigate the possible binding mode of the novel inhibitors with the aim of interpreting the biological data. Recently, we reported a study that identified two fundamental aspects of the interaction of 7-PPyQ derivatives with tubulin that enhanced our ability to obtain reliable docking results: which protein conformation is most suitable to accommodate the 7-PPyQ scaffold and which docking protocol performs best in reproducing the experimental structure of the complexes associated with that protein conformation [9]. Taking advantage of these findings, we docked eight of our derivatives, 19–24 and 29–30. As with the previously reported 7-PPyQ derivatives and, by analogy, with plinabulin [31], we observed that the most potent compounds 21, 23, and 24 occupied the colchicine site by establishing and maintaining a key hydrogen bond interaction with the backbone of βVal236 mediated by the pyrroloquinolinone scaffold. The fused-ring system, in addition, guaranteed strong hydrophobic interactions with βLeu253, βAla314, and βIle368. The substituents in position 7 established hydrophobic interactions with residues βPhe167, βTyr200, and βLeu250. The alkyl substituents at the N-pyrrole were placed in the pocket formed by residues βLys350, βThr351, βAla314, βAla352, and βThr179. Among the newly synthesized compounds, most of the small structural differences that caused strong divergences in activity are nicely explained by molecular docking (Video-S1). Compounds 21 and 22 only differ in the position of the conjugation of the naphthyl group in alpha and in beta position, respectively. While 21 preserves the typical interaction scheme, and, in particular, the hydrogen bond with βVal236, compound 22 fails to achieve a similar interaction (Fig. 7). More subtle structural differences, as with compounds 22–24, are more difficult to rationalize, even in terms of the shape of their substituent at position 7 (Fig. 7). While the most active derivative 24 is able, through the benzodioxole group to establish a further hydrogen bond with βGln134 in addition to the typical hydrogen mediated by the pyrroloquinolinone core with βVal236 (Fig. 7), both 22 and 23 fail to display the usual interaction with βVal236. The differences between the naphthyl and tetrahydronaphthyl substituents are minimal, but these differences abolish the activity of 22. However, it is difficult to ascribe the diverse biological effects to such small structural difference. Derivatives with a bulky substituent at the N-pyrrole apparently are able to retain the interaction pattern that occurs with the most potent compounds, 21, 23, and 24. Docking results suggest that such derivatives could theoretically be accommodated in the colchicine site and reproduce the binding mode of the most potent derivatives. The long linear alkyl chain (18 carbon atoms) characterizing analogs 19 and 20 may protrude between the beta-strand 373–381 and the alpha helix 252–259 and interact with the α-tubulin subunit. However, such a hydrophobic group would drastically affect the physicochemical properties of these compounds. The logP reaches values of 7.15 and 7.69 for 19 and 20, respectively (Table S1), suggesting a plausible sequestration by the membrane. Molecular docking also revealed that alternative scaffold geometries like the angular [3,2-h] PPyQ 29 and the [1,4]diazepin-indole derivative 30 were not able to reproduce the interaction pattern of the most potent compounds (per-residue interaction heat map profile, Video S1).

Fig. 7.

The energetically most favorable pose of compound 21–24 (magenta) obtained by molecular docking simulation using the protein conformation of the plinabulin complex (PDB ID: 5C8Y). The ribbon, as well as the residue atoms of the colchicine site, are colored according to the subunit to which they belong: white for β-tubulin and orange for α-tubulin. Hydrogen atoms are not shown. Hydrogen bonds are indicated in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supplementary video related to this article can be found at https://doi.org/10.1016/j.ejmech.2017.11.038.

3. Conclusion

Continuing our research aimed to expand our understanding of the SAR data of PPyQs, we synthesized a small library of analogs by chemically modifying three structural elements: substituents on the 3N pyrrole (19 and 20), varying the aryl group at the 7 position (21–24) and the [3,2-f] angular geometry (26–29). Evaluation of antiproliferative activity by the MTT test indicated that extensive elongation of the alkyl chain bound to 3N sharply decreased cytotoxicity (19 and 20), while enlargement of the aryl moiety at position 7 could increase cytotoxicity (21, 23 and 24). The unseparable mixture of compounds 26 and 27 showed that [f] and/or [g] angular geometries, respectively, could result in high cytotoxic activity, while compound 29, with [h] angular geometry did not. Compounds 21, 23 and 24 were the most cytotoxic compounds (nanomolar GI₅₀s) among the newly synthesized compounds, and they acted by an apoptotic mechanism and were excellent inhibitors of tubulin polymerization through an interaction at the colchicine site.

In addition, compounds 21 and 24 showed only modest effects in a rapidly proliferating noncancer cell and were completely inactive in quiescent cells. Both derivatives were not substrates for the Pgp drug efflux pump, as shown by their excellent cytotoxic activity against the multidrug-resistant CEM^Vbl.-100 cell line. Compound 24 also induced mitochondrial depolarization and ROS production, PARP activation and decreased expression of antiapoptotic proteins. The molecular docking studies of all derivatives showed that they could bind into the colchicine site in a manner similar to that of previously studied PPyQs [9]. Furthermore, the molecular docking studies provided good explanations for the effects of different substituents on the PPyQ nucleus. In particular, the molecular docking study revealed that the most active derivative 24 (sub-nanomolar GI₅₀s) is able through the benzodioxole group to establish an additional hydrogen bond with β-tubulin. Taking into account all of our results presented here, from a SAR point of view, compound 24 represents enhancement of the pharmacodynamics optimization process of the antimitotic PPyQ class, and it therefore merits in vivo evaluation.

4. Experimental section

4.1. Chemistry

Melting points were determined on a Buchi M-560 capillary melting point apparatus and are uncorrected. Infrared spectra were recorded on a PerkinElmer 1760 FTIR spectrometer with potassium bromide pressed disks; all values are expressed in cm− 1. UV−vis spectra were recorded on a Thermo Helyos α spectrometer. ¹H NMR spectra were determined on Bruker 300 and 400 MHz spectrometers, with the solvents indicated; chemical shifts are reported in δ (ppm) downfield from tetramethylsilane as internal reference. Coupling constants are given in hertz. In the case of multiplets, chemical shifts were measured starting from the approximate centre. Integrals were satisfactorily in line with those expected based on compound structure. Mass spectra were obtained on a Mat 112 Varian Mat Bremen (70 eV) mass spectrometer and Applied Biosystems Mariner System 5220 LC/MS (nozzle potential 140 eV). Column flash chromatography was performed on Merck silica gel (250–400 mesh ASTM); chemical reactions were monitored by analytical thin-layer chromatography (TLC) on Merck silica gel 60 F-254 glass plates. Microwave assisted reactions were performed on a CEM Discover^® monomode reactor with a built-in infrared sensor assisted-temperature monitoring and automatic power control; all reactions were performed in closed devices under pressure control. Solutions were concentrated on a rotary evaporator under reduced pressure. The purity of new tested compounds was checked by HPLC using the instrument HPLC VARIAN ProStar model 210, with detector DAD VARIAN ProStar 335. The analysis was performed with a flow of 1 mL/min, a C-18 column of dimensions 250 mm × 4.6 mm, a particle size of 5 mm, and a loop of 10 µL. The detector was set at 300 nm. The mobile phase consisted of phase A (Milli-Q H₂O, 18.0 MU, TFA 0.05%) and phase B (95% MeCN, 5% phase A). Gradient elution was performed as reported: 0 min, % B = 10; 20 min, % B = 90; 25 min, % B = 90; 26 min, % B = 10; 31 min, % B = 10.

Starting materials were purchased from Sigma-Aldrich and Alfa Aesar, and solvents were from Carlo Erba, Fluka and Lab-Scan. DMSO was obtained anhydrous by distillation under vacuum and stored over molecular sieves.

4.1.1. General procedure for the synthesis of 1N-Substituted nitroindoles (1–4)

As a typical procedure, the synthesis of 5-nitro-1-octadecyl-1H-indole 1 is described in detail. Into a two-necked 100 mL round-bottomed flask, 0.333 g (13.88 mmol) of NaH, 60% dispersion in mineral oil, was placed and washed with toluene (3 × 10 mL). With stirring, a solution of commercial 5-nitroindole, 0.750 g (4.625 mmol), in 5 mL of anhydrous DMF, was dropped into the flask, and the initial yellow color changed to red with the formation of H₂ gas. After 40 min at room temperature, the mixture was cooled to 0 °C, and 2.36 mL (6.94 mmol, d = 0.849 g/mL) of 1-chlorooctadecane was dropped into the flask and 0.050 g of NaI were added to the mixture. The reaction was monitored by TLC analysis (eluent toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, 25 mL of water was added, and the solvent was evaporated under reduced pressure, leaving a residue that was extracted with ethyl acetate (3 × 50 mL). The organic phase, washed with water and brine and dried over anhydrous Na₂SO₄, was concentrated under vacuum giving a crude yellow solid (2.522 g). This crude product was purified with a silica gel chromatographic column (d 3 cm, l 35 cm, 230–400 mesh, eluent n-hexane/toluene, 1:1), yielding 1.618 g of a pure yellow powder.

4.1.1.1. 5-nitro-1-octadecyl-1H-indole (1)

Yield: 84.5%; Rf: 0.48 (n-hexane/toluene, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 0.90 (t, J = 6.86 Hz, 3 H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.26–1.33 (m, 30 H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.87 (quin, J = 7.12 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 4.17 (t, J = 7.16 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 6.69 (dd, J = 3.22, 0.74 Hz, 1H, H-3), 7.26 (d, J = 3.24 Hz, 1H, H-2), 7.37 (d, J = 9.08 Hz, 1H, H-7), 8.13 (dd, J = 9.10, 2.22 Hz, 1H, H-6), 8.61 (d, J = 2.24 Hz, 1H, H-4) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 14.11 (N-CH₂CH₂(CH₂)₁₅CH₃), 22.68 (N-CH₂CH₂(CH₂)₁₅CH₃), 26.89 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.15 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.35 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.43 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.50 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.57 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.62 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.65 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.68 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.21 (N-CH₂CH₂(CH₂)₁₅CH₃), 31.92 (N-CH₂CH₂(CH₂)₁₅CH₃), 46.92 (N-CH₂CH₂(CH₂)₁₅CH₃), 103.85 (C-3), 109.18 (C-7), 117.13 (C-6), 118.27 (C-4), 127.69 (C-3a), 130.92 (C-2), 138.81 (C-7a), 141.50 (C-5) ppm.

4.1.1.2. 1-ethyl-5-nitro-1H-indole (2)

Compound 2 was prepared as for compound 1 by reacting commercial 5-nitroindole (1.50 g, 9.25 mmol), NaH (0.666 g, 27.75 mmol), and bromoethane (1.04 mL, 13.88 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis (eluent toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted to yield 1.649 g of yellow solid product. Yield: 93.7%; Rf: 0.67 (toluene/n-hexane/ethyl acetate, 1:1:1); ¹H NMR (300 MHz, DMSO-d₆): δ 0.55 (t, J = 7.1 Hz, 3H, CH₃), 3.47 (q, J = 7.1 Hz, 2H, CH₂), 5.93 (d, J = 3.3 Hz, 1H, H-2), 6.87 (m, 2H, H-7 e H-3), 7.20 (dd, J = 9.0, 2.1 Hz, 1H, H-6), 7.74 (d, J = 2.1 Hz, 1H, H-4) ppm.

4.1.1.3. 1-ethyl-6-nitro-1H-indole (3)

Compound 3 was prepared as for compound 1 by reacting commercial 6-nitroindole (1.00 g, 6.17 mmol), NaH (0.444 g, 18.50 mmol), and bromoethane (0.69 mL, 9.25 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis (eluent toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted to yield 1.110 g of yellow solid. Yield: 94.5%; Rf: 0.75 (toluene/n-hexane/ethyl, 1:1:1); ¹H NMR (300 MHz, CDCl₃): δ 1.45 (t, J = 7.29 Hz, 3H, CH₂CH₃), 4.20 (q, J = 7.30 Hz, 2H, CH₂CH₃), 6.52 (dd, J = 3.07 Hz e J = 0.82 Hz, 1H, H-3), 7.34 (d, J = 3.09 Hz, 1H, H-2), 7.58 (d, J = 8.73 Hz, 1H, H-4), 7.93 (dd, J = 8.79 Hz e J = 2.04 Hz, 1H, H-5), 8.27 ppm (d, J = 1.95 Hz, 1H, H-7); ¹³C NMR (75 MHz, CDCl₃): δ 15.60 (CH₂CH₃), 41.53 (CH₂CH₃), 102.28 (C-3), 106.40 (C-7), 114.85 (C-5), 120.80 (C-4), 132.83 (C-2), 133.41 (C-3a), 134.27 (C-7a), 142.85 (C-6) ppm.

4.1.1.4. 1-ethyl-7-nitro-1H-indole (4)

Compound 4 was prepared as for compound 1 by reacting commercial 7-nitroindole (1.00 g, 6.17 mmol), NaH (0.444 g, 18.50 mmol), and bromoethane (0.69 mL, 9.25 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis (eluent toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted to yield 1.103 g of yellow solid. Yield: 94.0%; Rf: 0.82 (toluene/n-hexane/ethyl acetate, 1:1:1); ¹H NMR (300 MHz, CDCl₃): δ 1.33 (t, J = 7.23 Hz, 3H, CH₂CH₃), 4.30 (q, J = 7.21 Hz, 2H, CH₂CH₃), 6.67 (d, J = 3.24 Hz, 1H, H-3), 7.14 (t, J = 7.84 Hz, 1H, H-5), 7.22 (d, J = 3.24 Hz, 1H, H-2), 7.82 (dd, J = 7.84, 0.82 Hz, 1H, H-4), 7.87 (dd, J = 7.78, 1.00 Hz, 1H, H-6) ppm; ¹³C NMR (75 MHz CDCl₃): δ 16.01 (CH₂CH₃), 44.83 (CH₂CH₃), 103.01 (C-3), 118.40 (C-5), 119.78 (C-4), 126.13 (C-3a), 127.13 (C-6), 132.00 (C-2), 133.83 (C-7a), 136.87 (C-7) ppm.

4.1.2. General procedure for the synthesis of 1N-Substituted nitroindoles (5–8)

As a typical procedure, the synthesis of 1-octadecyl-1H-indol-5-amine 5 is described in detail. Into a two-necked flask, previously dried in an oven, about 0.300 g of Pd/C 10% and approximately 50 mL of ethyl acetate were placed. After connecting the flask to an elastomer balloon containing H₂, the mixture was stirred at room temperature for 1 h in order to saturate the suspension of Pd/C with H₂. Then, 1.00 g (2.41 mmol) of nitroindole derivative 1 in 15 mL of ethyl acetate was added dropwise to the suspension, and the mixture was stirred under H₂ at atmospheric pressure and heated by means of an oil bath at 50–60 °C, monitoring the progress of the reaction by TLC analysis (toluene/n-hexane, 1:1). At the end of the reaction, the mixture was filtered through a celite pad, and the solution was concentrated under vacuum to give 0.784 g of semi-solid dark purple product.

4.1.2.1. 1-octadecyl-1H-indol-5-amine (5)

Yield: 84.8%; Rf: 0.12 (n-hexane:toluene, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 0.90 (t, J = 6.78 Hz, 3H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.26–1.31 (m, 30H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.82 (quin, J = 7.06 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 4.05 (t, J = 7.12 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 6.31 (d, J = 3.04 Hz, 1H, H-3), 6.72 (dd, J = 8.58, 2.22 Hz, 1H, H-6), 6.98 (d, J = 2.16 Hz, 1H, H-4), 7.03 (d, J = 3.04 Hz, 1H, H-2), 7.17 ppm (d, J = 8.60 Hz, 1H, H-7); ¹³C NMR (101 MHz, CDCl3): δ 14.11 (N-CH₂CH₂(CH₂)₁₅CH₃), 22.69 (N-CH₂CH₂(CH₂)₁₅CH₃), 27.01 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.26 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.36 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.50 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.57 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.61 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.64 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.66 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.67 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.69 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.26 (N-CH₂CH₂(CH₂)₁₅CH₃), 31.93 (N-CH₂CH₂(CH₂)₁₅CH₃), 46.50 (N-CH₂CH₂(CH₂)₁₅CH₃), 99.52 (C-3), 106.21 (C-4), 109.93 (C-7), 112.48 (C-6), 128.21 (C-2), 129.39 (C-3a), 131.29 (C-7a), 138.36 (C-5) ppm.

4.1.2.2. 1-ethyl-1H-indol-5-amine (6)

Compound 6 was prepared as for compound 5 by reacting 1.65 g of compound 2 (8.67 mmol), obtaining 1.36 g of a semisolid purple compound. Yield: 97.9%; Rf: 0.28 (toluene/n-hexane/ethyl, 1:1:1); ¹H NMR (300 MHz, DMSO-d₆): δ 1.55 (t, J = 7.3 Hz, 3H, CH₃), 4.06 (q, J = 7.3 Hz, 2H, CH₂), 4.48 (s br, 2H, NH₂), 6.15 (d, J = 3.05 Hz, 1H, H-2), 6.54 (dd, J = 8.5, 2.28 Hz, 1H, H-6), 6.69 (d, J = 2.28 Hz, 1H, H-4), 7.16 (m, 2H, H-3 e H-7) ppm.

4.1.2.3. 1-ethyl-1H-indol-6-amine (7)

Compound 7 was prepared as for compound 5 by reacting 1.45 g of compound 3 (7.61 mmol), obtaining 1.26 g of a semisolid purple compound. Yield: 99.8%; Rf: 0.28 (toluene/n-hexane/ethyl acetate, 1:1:1); ¹H NMR (300 MHz, CDCl₃): δ1.33 (t, J = 7.26 Hz, 3H, CH₂CH₃), 3.42 (s, 2H, NH₂), 3.95 (q, J = 7.26 Hz, 2H, CH₂CH₃), 6.28 (d, J = 3.00 Hz, 1H, H-3), 6.47 (dd, J = 8.29, 2.95 Hz, 1H, H-5), 6.54 (d, J = 1.94 Hz, 1H, H-7), 6.83 (d, J = 3.29 Hz, 1H, H-2), 7.31 ppm (d, J = 8.31 Hz, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃): δ 15.30 (CH₂CH₃), 40.80 (CH₂CH₃), 94.95 (C-7), 100.94 (C-3), 110.29 (C-5), 121.57 (C-4), 122.03 (C-3a), 125.14 (C-2), 136.91 (C-7a), 141.69 (C-6) ppm.

4.1.2.4. 1-ethyl-1H-indol-7-amine (8)

Compound 8 was prepared as for compound 5 by reacting 1.43 g of compound 3 (7.64 mmol), obtaining 1.34 g of a semisolid purple compound. Yield: 99.8%; Rf: 0.48 (toluene/n-hexane/ethyl acetate, 1:1:1); ¹H NMR (300 MHz, CDCl₃): δ 1.51 (t, J = 7.23 Hz, 3H, CH₂CH₃), 3.72 (s, 2H, NH₂), 4.43 (q, J = 7.21 Hz, 2H, CH₂CH₃), 6.45 (d, J = 3.09 Hz, 1H, H-3), 6.53 (dd, J = 7.36, 0.88 Hz, 1H, H-4), 6.93 (dd, J = 7.83, 7.44 Hz, 1H, H-5), 7.01 (d, J = 3.12 Hz, 1H, H-2), 7.17 ppm (dd, J = 7.92, 1.05 Hz, 1H, H-6); ¹³C NMR (75 MHz CDCl₃): δ 18.13 (CH₂CH₃), 43.87 (CH₂CH₃), 101.64 (C-3), 110.20 (C-4), 113.23 (C-6), 120.19 (C-5), 126.52 (C-3a), 128.74 (C-2), 131.19 (C-7a), 132.14 (C-7) ppm.

4.1.3. General procedure for the synthesis of aryl ethyl acetate derivatives 9–12

As a typical procedure, the preparation of aryl ethyl acetate derivative 9 is described in detail. 2.36 g (98.33 mmol) of NaH 60% dispersion in mineral oil was placed in a 100 mL round-bottomed flask and washed with toluene to remove oil. Then, 7.12 mL (59.75 mmol, d = 0.975 g/mL) of diethyl carbonate and 20 mL of anhydrous dioxane were added. The mixture was heated to 80 °C with stirring and then 4.46 mL (29.37 mmol, d = 1.120 g/mL) of 1-acetyl naphthalene was added dropwise. The mixture was stirred at 80 °C for 15 h and monitored by TLC analysis (eluent ethyl acetate/petroleum ether, 1:5). The reaction mixture was allowed to cool to room temperature, and 25 mL of water was added. The water layer was separated and extracted twice with ethyl acetate. The organic layers were combined and washed with water and brine, dried over Na₂SO₄, filtered, and evaporated to dryness, giving 5.73 g of a yellow oil. The crude product was used for the next reaction step without further purification.

4.1.3.1. Ethyl 3-(naphthalen-5-yl)-3-oxopropanoate (9)

Yield: 80.52%; Rf: 0.68 (EtOAc:petroleum ether, 1:5); ¹H-NMR (DMSO-d₆) = δ 1.11 (t, J = 7.09 Hz, 3H, OCH₂CH₃), 4.08 (q, J = 7.09 Hz, 2H, OCH₂CH₃), 4.30 (s, 2H, COCH₂CO), 7.68 (m, 3H, H-3, H-6, H-7), 8.03 (m, J = 8.84 Hz, 1H, H-8), 8.18 (m, J = 8.23 Hz, 2H, H-4, H-5), 8.57 (d, J = 8.39 Hz, 1H, H-2).

4.1.3.2. Ethyl 3-(naphthalen-6-yl)-3-oxopropanoate (10)

Compound 10 was prepared as for compound 9 by reacting 2.5 g of commercial 2-acetyl-naphthalene (14.69 mmol) with 3.55 mL (29.31 mmol) of diethyl carbonate and 1.17 g (48.79 mmol) of NaH in 20 mL of dioxane to yield 2.45 g of crude product. This was purified by column chromatography (d 3 cm, l 35 cm, 230–400 mesh, eluent ethyl acetate/petroleum ether, 1:5), yielding 1.429 g of a yellow solid. Yield: 40.1%; Rf: 0.55 (EtOAc:petroleum ether, 1:5); ¹H NMR (300 MHz, CDCl₃): δ 1.19 (t, J = 7.14 Hz, 3H, OCH₂CH₃) 4.05 (s, 2H, OCH₂O), 4.16 (q, J = 7.13 Hz, 2H, OCH₂CH₃), 7.49–7.58 (m, 2H, H-6′ e H-7′), 7.81 (d, J = 8.58 Hz, 1H, H-5′), 7.84 (d, J = 8.49 Hz, 1H, H-4′), 7.90 (d, J = 7.98 Hz, 1H, H-8′), 7.94 (dd, J = 8.68, 1.78 Hz, 1H, H-3′), 8.38 (d, J = 1.26 Hz, 1H, H-1′). Keto-enol ratio 81:19.

4.1.3.3. Ethyl 3-(1, 2, 3, 4-tetrahydronaphthalen-7-yl)-3-oxopropanoate (11)

Compound 11 was prepared as for compound 9 by reacting 0.945 mL of commercial 6-acetyl-1, 2, 3, 4-tetrahydronaphthalene (5.74 mmol) with 1.39 mL (11.48 mmol) of diethyl carbonate and 0.457 g (19.06 mmol) of NaH in 20 mL of dioxane to yield 0.694 g of crude product. This was purified by column chromatography (d 3 cm, l 35 cm, 230–400 mesh, eluent ethyl acetate/petroleum ether, 1:5), yielding 0.351 g of a yellow solid. Yield: 24.8%; Rf: 0.78 (EtOAc:petroleum ether, 1:5); ¹H NMR (300 MHz, CDCl₃): δ 1.25 (t, J = 7.15 Hz, 3H, OCH₂CH₃), 1.81 (m, 4H, H-6′ e H-7′), 2.81 (m, 4H, H-5′ e H-8′), 3.94 (s, 2H, OCH₂O), 4.20 (q, J = 7.13 Hz, 2H, OCH₂CH₃), 7.14 (d, J = 8.61 Hz, 1H, H-4′), 7.63 (dd, J = 7.18, 1.87 Hz, 1H, H-3′), 7.65 (m, 1H, H-1′). Keto-enol ratio 90:10.

4.1.3.4. Ethyl 3-(benzo[d][1, 3]dioxol-6-yl)-3-oxopropanoate (12)

Compound 12 was prepared as for compound 9 by reacting 1.642 g of commercial 1-(benzo[d][1, 3]dioxol-6-yl)ethanone (10 mmol) with 2.42 mL (20 mmol) of diethyl carbonate and 0.672 g (28 mmol) of NaH in 20 mL of THF to yield 2.162 g of a yellow oil. The crude product is used for the next reaction step without further purification. Yield: 91.5%; Rf: 0.40 (EtOAc:petroleum ether, 1:5); ¹H NMR (300 MHz, CDCl₃): δ 1.25 (t, J = 7.14 Hz, 3H, OCH₂CH₃), 3.90 (OCCH₂CO), 4.19 (q, J = 7.14 Hz, 2H, OCH₂CH₃), 6.04 (COCH₂OC), 6.84 (d, J = 8.16 Hz, 1H, H-4′), 7.41 (d, J = 1.74 Hz, 1H, H-1′), 7.51 (dd, J = 8.17, 1.78 Hz, 1H, H-3′). Keto-enol ratio 95:5.

4.1.4. General procedure for the synthesis of acrylate derivatives 13–18, 25 and 28

As a typical procedure, the synthesis of acrylate derivative 13 is described in detail. In a 100 mL round-bottomed flask, 0.786 g (2.045 mmol) of 3-substituted aminoindole 5 in 10 mL of absolute ethanol was condensed with 0.388 mL (3.07 mmol; d = 1.029 g/mL) of commercial ethyl acetoacetate and 0.5 mL of glacial acetic acid in the presence of 100 mg of drierite. The mixture was refluxed for about 48 h, the reaction being monitored by TLC analysis (n-hexane/ethyl acetate, 8:2). At the end of the reaction, the mixture was cooled and filtered to remove the drierite. The resulting solution was evaporated to dryness under vacuum and the residue (1.039 g) purified by silica gel chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate, 8:2) to yield 0.770 g of a semisolid brown product.

4.1.4.1. (E, Z)-ethyl 3-(1-octadecyl-1H-indol-5-ylamino)but-2-enoate (13)

Yield: 75.8%; Rf: 0.80 (n-hexane/ethyl acetate, 8:2); ¹H NMR (400 MHz, CDCl₃): δ 0.90 (t, J = 6.85 Hz, 3H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.27–1.33 (m, 30H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.31 (t, J = 7.10 Hz, 3H, COOCH₂CH₃), 1.85 (quin, J = 6.88 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.93 (s, 3H, NHCCH₃) 4.11 (t, J = 7.20 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 4.18 (q, J = 7.13 Hz, 2H, COOCH₂CH₃), 4.67 (s, 1H, NHCCH), 6.46 (d, J = 3.08 Hz, 1H, H-3), 6.97 (dd, J = 8.56, 1.96 Hz, 1H, H-6), 7.13 (d, J = 3.12 Hz, 1H, H-2), 7.28 (d, J = 8.56 Hz, 1H, H-7), 7.37 (d, J = 1.92 Hz, 1H, H-4) 10.28 ppm (s, 1H, NH); ¹³C NMR (101 MHz, CDCl₃): δ 14.11 (N-CH₂CH₂(CH₂)₁₅CH₃), 14.67 (COOCH₂CH₃), 20.25 (NHCCH₃) 20.25 (N-CH₂CH₂(CH₂)₁₅CH₃), 22.68 (N-CH₂CH₂(CH₂)₁₅CH₃), 27.00 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.23 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.25 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.35 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.49 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.56 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.61 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.65 (NCH₂CH₂(CH₂)₁₅CH₃), 29.67 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.69 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.11 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.26 (N-CH₂CH₂(CH₂)₁₅CH₃), 31.92 (NCH₂CH₂(CH₂)₁₅CH₃), 46.62 (N-CH₂CH₂(CH₂)₁₅CH₃), 58.50 (COOCH₂CH₃), 83.81 (NHCCH), 100.89 (C-3), 109.51 (C-7), 118.05 (C-4), 120.38 (C-6), 128.61 (C-3a), 128.83 (C-2), 131.16 (C-7a), 134.21 (C-5), 160.94 (NHCCH₃), 170.57 (COOCH₂CH₃) ppm.

4.1.4.2. (E, Z)-ethyl 3-(1-octadecyl-1H-indol-5-ylamino)-3-phenylacrylate (14)

Compound 14 was prepared as for compound 13 by reacting 0.974 mL of commercial ethyl benzoylacetate (5.07 mmol, d = 1.11 g/mL) with 1.301 g (3.38 mmol) of previously prepared compound 5, yielding 2.328 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate, 8:2) to yield 0.405 g of a semisolid brown product. Yield: 21.7%; Rf: 0.77 (n-hexane/ethyl acetate, 8:2); ¹H NMR (300 MHz, CDCl₃): δ 0.80 (t, J = 6.70 Hz, 3H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.18–1.16 (m, 30H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.24 (t, J = 7.10 Hz, 3H, COOCH₂CH₃) 1.68 (quin, J = 6.75 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 3.91 (t, J = 7.20 Hz, 2H, NCH₂CH₂(CH₂)₁₅CH₃), 4.13 (q, J = 7.11 Hz, 2H, COOCH₂CH₃), 4.85 (s, 1H, NHCCH), 6.19 (d, J = 3.03 Hz, 1H, H-3), 6.53 (dd, J = 8.68, 2.11 Hz, 1H, H-6), 6.92 (d, J = 1.72 Hz, 1H, H-4), 6.93 (d, J = 3.12 Hz, 1H, H-2), 6.95 (d, J = 9.00 Hz, 1H, H-7), 7.13 (m, 1H, H-4′), 7.16 (m, 2H, H-3′ e H-5′), 7.28 (m, 2H, H-2′ e H-6′), 10.15 (s, 1H, NH) ppm. ¹³C NMR (75 MHz, CDCl₃): δ 14.10 (N-CH₂CH₂(CH₂)₁₅CH₃), 14.67 (COOCH₂CH₃), 20.22 (N-CH₂CH₂(CH₂)₁₅CH₃), 22.98 (N-CH₂CH₂(CH₂)₁₅CH₃), 27.06 (NCH₂CH₂(CH₂)₁₅CH₃), 29.12 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.34 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.56 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.67 (NCH₂CH₂(CH₂)₁₅CH₃), 29.76 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.82 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.84 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.91 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.23 (NCH₂CH₂(CH₂)₁₅CH₃), 30.67 (N-CH₂CH₂(CH₂)₁₅CH₃), 31.45 (N-CH₂CH₂(CH₂)₁₅CH₃), 46.16 (N-CH₂CH₂(CH₂)₁₅CH₃), 57.27 (COOCH₂CH₃), 84.18 (NHCCH), 104.28 (C-3), 113.28 (C-4), 114.27 (C-7), 116.28 (C-6), 124.82 (C-3a), 127.82 (C-2), 128.36 (C-2′ e C-6′), 129.27 (C-3′ e C-5′) 130.17 (C-4′), 130.28 (C-7a), 131.01 (C-1′), 139.82 (C-5), 154.85 (NHCCH), 173.82 (COOCH₂CH₃) ppm.

4.1.4.3. (E, Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(naphthalen-1-yl)acrylate (15)

Compound 15 was prepared as for compound 13 by reacting 1.815 g (7.49 mmol) of compound 9 with 1 g (6.24 mmol) of previously prepared compound 6, giving 2.461 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate/toluene, 1:1:1) to yield 0.879 g of a semisolid brown product. Yield: 36.6%; Rf: 0.82 (n-/acetate, 8:2); ¹H NMR (400 MHz, DMSO-d₆): δ 1.21 (t, J = 7.16 Hz, 3H, COOCH₂CH₃), 1.41 (t, J = 7.20 Hz, 3H, NCH₂CH₃), 4.19 (q, J = 7.14 Hz, 2H, COOCH₂CH₃), 4.35 (q, J = 7.22 Hz, 2H, NCH₂CH₃), 4.92 (s, 1H, NHCCH), 7.24 (d, J = 2.12 Hz, 1H, H-5), 7.43 (dd, J = 8.81 Hz, 1H, H-6), 7.59 (m, 1H, H-3), 7.85 (m, 1H, H-2), 7.90 (dd, J = 8.95, 0.64 Hz, 1H, H-7), 8.08 (m, J = 8.56 Hz e J = 2.14 Hz, 1H, H-8′), 8.13 (m, J = 8.23 Hz, 1H, H-2′), 11.93 ppm (s, 1H, NH); ¹³C NMR (101 MHz, DMSO-d₆): δ 16.39 (NHCH₂CH₃), 17.04 (COOCH₂CH₃), 41.03 (NCH₂CH₃), 60.04 (COOCH₂CH₃), 88.94 (NHCCH), 104.10 (C-3), 112.53 (C-6), 115.98 (C-7), 118.31 (C-4), 123.64 (C-3a), 125.30 (C-2), 125.87 (C-aro), 126.94 (Caro), 127.64 (C-aro), 127.77 (C-aro), 128.64 (C-aro), 128.92 (C-8′), 130.12 (C-2′), 131.06 (C-8′ a), 131.46 (C-4′), 132.44 (C-7a), 133.61 (C-1′), 147.37 (C-5), 153.12 (NHCCH), 170.24 (CO).

4.1.4.4. (E, Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(naphthalen-3-yl)acrylate (16)

16 was prepared as for compound 13 by reacting 1 g (4.13 mmol) of compound 10 with 0.440 g (2.75 mmol) of previously prepared compound 6, giving 1.567 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-/ethyl acetate 8:2) to yield 0.692 g of a semisolid orange product. Yield: 65.5%; Rf: 0.44 (n-hexane/ethyl acetate, 8:2); ¹H NMR (300 MHz, CDCl₃): δ 1.19 (t, J = 7.12 Hz, 3H, NCH₂CH₃), 1.30 (t, J = 7.26 Hz, 3H, COOCH₂CH₃), 3.95 (q, J = 7.27 Hz, 2H, NCH₂CH₃), 4.16 (q, J = 7.12 Hz, 2H, COOCH₂CH₃), 4.98 (s, 1H, NHCCH), 6.17 (dd, J = 3.09, 0.51 Hz, 1H, H-3), 6.58 (dd, J = 8.73, 2.07 Hz, 1H, H-6), 7.00 (dd, J = 1.47 Hz, 1H, H-4), 7.24 (dd, J = 8.53, 1.69 Hz, 1H, H-3′), 7.38 (m, 1H, H-7′), 7.39 (d, J = 2.97 Hz, 1H, H-2), 7.46 (m, 1H, H-6), 7.84 (d, J = 8.49 Hz, 1H, H-7), 7.92 (m, 1H, H-5′), 7.93 (m, 1H, H-8′), 8.38 (s, 1H, H-1′), 10.39 (s, 1H, NH) ppm. ¹³C NMR (75 MHz, CDCl₃): δ 15.92 (NCH₂CH₃), 16.38 (COOCH₂CH₃), 41.38 (NCH₂CH₃), 59.27 (COOCH₂CH₃), 90.28 (NHCCH), 101.27 (C-3), 109.27 (C-4), 115.88 (C-7), 116.27 (C-6), 126.93 (C-5′), 126.95 (C-6′), 127.27 (C-3′), 127.37 (C-2), 127.82 (C-7′), 127.82 (C-8′), 129.73 (C-4′), 129.81 (C-3a), 132.81 (C-7a), 133.82 (C-8′ a), 133.98 (C-1′), 134.82 (C-4′ a), 135.62 (C-2′), 142.73 (C-5), 149.12 (NHCCH), 175.98 (CO) ppm.

4.1.4.5. (E, Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(1, 2, 3, 4-tetrahydronaphthalen-7-yl)acrylate (17)

Compound 17 was prepared as for compound 13 by reacting 0.373 g (1.51 mmol) of compound 11 with 0.201 g (1.26 mmol) of previously prepared compound 6, giving 0.535 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate 8:2) to yield 0.337 g of a semisolid brown-orange product. Yield: 68.8%; Rf: 0.54 (n-hexane/ethyl acetate, 8:2); ¹H NMR (300 MHz, CDCl₃): δ 1.19 (t, J = 7.14 Hz, 3H, NCH₂CH₃), 1.34 (t, J = 7.27 Hz, 3H, COOCH₂CH₃), 1.67 (m, 4H, H-6′ e H-7′), 2.60 (m, 4H, H-5′ e H-8′), 4.00 (q, J = 7.28 Hz, 2H, NCH₂CH₃), 4.13 (q, J = 7.11 Hz, 2H, COOCH₂CH₃), 4.83 (s, 1H, NHCCH), 6.23 (dd, J = 3.07, 0.58 Hz, 1H, H-3), 6.57 (dd, J = 8.67, 2.10 Hz, 1H, H-6), 6.76 (d, J = 7.95 Hz, 1H, H-7), 6.97 (d, J = 2.91 Hz, 1H, H-1′), 7.07 (d, J = 8.55 Hz, 1H, H-3′), 7.42 (d, J = 1.56 Hz, 1H, H-4′), 7.57 (m, 1H, H-4′), 7.57 (m, 1H, H-2), 10.27 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): d 15.92 (NCH₂CH₃), 15.93 (COOCH₂CH₃), 24.96 (C-6′ e C-7′), 30.04 (C-5′ e C-8′), 40.72 (NCH₂CH₃), 61.38 (COOCH₂CH₃), 89.97 (NHCCH), 102.83 (C-3), 108.38 (C-4), 116.23 (C-6), 117.28 (C-7), 124.28 (C-3a), 124.82 (C-3′), 126.28 (C-1′), 127.28 (C-4′), 127.73 (C-2), 132.73 (C-7a), 135.28 (C-4′ a), 136.93 (C-8′ a), 139.83 (C-1′), 142.82 (C-5), 156.38 (NHCCH), 175.81 (CO) ppm.

4.1.4.6. (E, Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(benzo[d][1, 3]dioxol-6-yl)acrylate (18)

Compound 18 was prepared as for compound 13 by reacting 1.130 g (4.79 mmol) of compound 12 with 0.639 g (3.99 mmol) of previously prepared compound 6, giving 1.761 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate 8:2) to yield 0.558 g of a semisolid brown product. Yield: 16.1%; Rf: 0.67 (n-hexane/ethyl acetate, 8:2); ¹H NMR (300 MHz, CDCl₃): δ 1.26 (t, J = 7.18 Hz, 3H, NCH₂CH₃), 1.37 (t, J = 7.23 Hz, 3H, COOCH₂CH₃), 4.08 (q, J = 7.28 Hz, 2H, NCH₂CH₃), 4.33 (q, J = 7.13 Hz, 2H, COOCH₂CH₃), 4.89 (s, 1H, NHCCH), 6.05 (s, 2H, OCH₂O), 6.31 (d, J = 3.09 Hz, 1H, H-3), 6.65 (d, J = 8.10 Hz, 1H, H-7), 6.86 (d, J = 8.19 Hz, 1H, H-4′), 6.89 (dd, J = 8.09, 1.71 Hz, 1H, H-6), 7.02 (d, J = 2.01 Hz, 1H, H-4), 7.05 (d, J = 3.15 Hz, 1H, H-2), 7.43 (d, J = 1.69 Hz, 1H, H-1′), 7.53 (dd, J = 8.17, 1.78 Hz, 1H, H-3′), 10.28 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 15.93 (NCH₂CH₃), 16.38 (COOCH₂CH₃), 41.28 (NCH₂CH₃), 61.28 (COOCH₂CH₃), 90.03 (NHCCH), 100.24 (OCH₂O), 104.28 (C-3), 108.28 (C-4), 110.28 (C-4′), 115.38 (C-7), 116.28 (C-6), 122.36 (C-3), 122.38 (C-3a), 124.73 (C-1), 127.82 (C-2), 135.47 (C-1′), 136.38 (C-7a), 142.38 (C-5), 147.28 (C-4′b), 148.37 (C-4a'), 157.38 (NHCCH), 174.28 (CO) ppm.

4.1.4.7. (E, Z)-ethyl 3-(1-ethyl-1H-indol-6-ylamino)-3-phenylacrylate (25)

Compound 25 was prepared as for compound 13 by reacting 2.27 g of commercial ethyl benzoylacetate (11.79 mmol, d = 1.11 g/mL) with 1.26 g (7.86 mmol) of previously prepared compound 7, giving 2.943 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate 6:4) to yield 2.069 g of a brown product. Yield: 78.7%; Rf: 0.87 (n-hexane/ethyl acetate, 6:4); ¹H NMR (300 MHz, CDCl₃): δ 1.10 (t, J = 7.26 Hz, 3H, NCH₂CH₃), 1.25 (t, J = 7.12 Hz, 3H, COOCH₂CH₃), 3.78 (q, J = 7.27 Hz, 2H, NCH₂CH₃), 4.14 (q, J = 7.11 Hz, 2H, COOCH₂CH₃), 4.89 (s, 1H, NHCCH) 6.26 (dd, J = 3.12, 0.75 Hz, 1H, H-3), 6.45 (d, J = 1.30 Hz, 1H, H-7), 6.51 (dd, J = 8.40, 1.92 Hz, 1H, H-5), 6.88 (d, J = 3.12 Hz, 1H, H-2), 7.16 (m, 1H, H-4′), 7.19 (m, 2H, H-3′ e H-5′), 7.26 (d, J = 8.37 Hz, 1H, H-4), 7.30 (m, J = 8.77 Hz, 2H, H-2′ e H-6′), 10.39 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 14.61 (NCH₂CH₃), 15.10 (COOCH₂CH₃), 40.91 (NCH₂CH₃), 59.19 (COOCH₂CH₃), 89.51 (NHCCH), 100.74 (C-3), 103.76 (C-7), 115.86 (C-5), 120.77 (C-4), 124.87 (C-3a), 127.10 (C-2), 128.29 (C-2′ e C-6′), 128.39 (C-3′ e C-5′), 129.11 (C-4′), 134.72 (C-1′), 135.51 (C-7a), 136.45 (C-6), 159.84 (NHCCH), 170.34 (CO) ppm.

4.1.4.8. (E, Z)-ethyl 3-(1-ethyl-1H-indol-7-ylamino)-3-phenylacrylate (28)

Compound 28 was prepared as for compound 13 by reacting 2.418 g of commercial ethyl benzoylacetate (12.59 mmol, d = 1.11 g/mL) with 1.35 g (8.39 mmol) of previously prepared compound 8, giving 3.166 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent n-hexane/ethyl acetate 6:4) to yield 1.242 g of a brown product. Yield: 44.3%; Rf: 0.84 (n-hexane/ethyl acetate, 6:4); ¹H NMR (300 MHz, CDCl₃): δ 1.28 (t, J = 7.14 Hz, 3H, NCH₂CH₃), 1.50 (q, J = 7.15 Hz, 2H, COOCH₂CH₃), 4.24 (q, J = 7.15 Hz, 2H, COOCH₂CH₃), 4.42 (q, J = 7.23 Hz, 2H, NCH₂CH₃), 5.13 (s, 1H, NHCCH), 6.44 (d, J = 3.12 Hz, 1H, H-3), 6.52 (dd, J = 7.35, 0.72 Hz, 1H, H-4), 6.91 (t, J = 7.63 Hz, 1H, H-5), 7.00 (d, J = 3.12 Hz, 1H, H-2), 7.16 (dd, J = 7.92, 0.87 Hz, 1H, H-6), 7.51 (m, J = 7.80 Hz, 2H, H-3′ e H-5′), 7.62 (m, J = 7.46, 2.05 Hz, 1H, H-4′), 7.97 (m, J = 8.55 Hz, 2H, H-2′ e H-6′), 10.62 ppm (s, 1H, NH); ¹³C NMR (75 MHz, CDCl₃): δ 14.09 (NCH₂CH₃), 18.11 (COOCH₂CH₃), 43.86 (NCH₂CH₃), 61.52 (COOCH₂CH₃), 90.31 (NHCCH), 101.63 (C-3), 110.19 (C-4), 120.18 (C-5) 126.06 (C-3a), 128.52 (C-6) 128.56 (C-2), 128.72 (C-4′), 128.72 (C-6), 128.80 (C-3′ e C-5′), 133.78 (C-1′), 135.94 (C-7a), 135.99 (C-7), 161.15 (NHCCH), 173.23 (CO) ppm.

4.1.5. General procedure for the synthesis of phenylpyrroloquinolinones 19–24, 26, 27 and 29

As a typical procedure, the synthesis of the phenylpyrroloquinolinone derivative 19 is described in detail. In a two-necked round-bottomed flask, 30 mL of diphenyl ether was heated to boiling. Acrylate derivative 13 (0.758 g, 1.53 mmol) was then added portionwise, and the resulting mixture was refluxed for 15 min. After cooling to room temperature, 25 mL of diethyl ether was added, and the mixture was left for 12 h. The separated precipitate was collected by filtration and washed many times with diethyl ether. The crude product (0.534 g) was purified by flash column chromatography (eluent CHCl₃/methanol, 9:1), obtaining 0.165 g of a white powder.

4.1.5.1. 7-methyl-3-octadecyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (19)

Yield: 23.6%; Rf: 0.59 (blue fluorescent spot, CHCl₃/methanol, 9:1); mp = 140 °C; ¹H NMR (400 MHz, CDCl₃): δ 0.89 (t, J = 6.84 Hz, 3H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.31–1.25 (m, 30H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.86 (quin, J = 6.64 Hz, 2H, NCH₂CH₂(CH₂)₁₅CH₃), 2.45 (s, 3H, NHCCH₃), 4.21 (t, J = 7.06 Hz, 2H, NCH₂CH₂(CH₂)₁₅CH₃), 6.39 (s, 1H, H-8), 7.28 (d, J = 2.90 Hz, 1H, H-2), 7.63 (d, J = 9.00 Hz, 1H, H-4), 7.69 (d, J = 8.92 Hz, 1H, H-5), 7.83 (d, J = 2.88 Hz, 1H, H-1), 12.22 (s, 1H, NH) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 14.00 (N-CH₂CH₂(CH₂)₁₅CH₃), 19.67 (NHCCH₃), 22.48 (N-CH₂CH₂(CH₂)₁₅CH₃), 24.99 (N-CH₂CH₂(CH₂)₁₅CH₃), 26.74 (NCH₂CH₂(CH₂)₁₅CH₃), 29.03 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.14 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.29 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.35 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.40 (NCH₂CH₂(CH₂)₁₅CH₃), 29.44 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.48 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.56 (N-CH₂CH₂(CH₂)₁₅CH₃), 31.71 (N-CH₂CH₂(CH₂)₁₅CH₃), 46.53 (NCH₂CH₂(CH₂)₁₅CH₃), 104.29 (C-1), 108.73 (C-8), 111.88 (C-5), 115.48 (C-4), 116.69 (C-9a), 122.92 (C-9b), 128.32 (C-2), 131.55 (C-3a), 136.44 (C-5a), 147.80 (C-7), 182.01 (C-9) ppm; IR (KBr): ν = 3391.59 (NH), 2919.43 (aliphatic C-H), 2847.80 (aliphatic C-H), 1637.16 (C==O), 1522.64 (C==C) cm⁻¹; UV-Vis (H₂O): 231.18 nm (A = 203.78 mAU), 275.70 nm (A = 93.22 mAU), 351.08 nm (45.03 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 416 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₃₀H₄₇N₂O⁺, 451.3688; found, 451.3769; RP-C18 HPLC: t_R = 26.96 min, 99.9%.

4.1.5.2. 3-octadecyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (20)

Compound 20 was prepared as described for compound 19 by reacting 0.406 g (7.3 mmol) of the appropriate phenylacrylate derivative 14 to yield 0.389 g of a raw solid, which was purified by flash column chromatography (eluent CHCl₃/methanol, 95:5) to yield 0.175 g of a grey powder. Yield: 47.1%; Rf: 0.38 (blue fluorescent spot, CHCl₃/methanol, 95:5); mp = 156 °C; ¹H NMR (300 MHz, CDCl₃): δ 0.89 (t, J = 6.72 Hz, 3H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.25–1.32 (m, 30H, N-CH₂CH₂(CH₂)₁₅CH₃), 1.88 (quin, J = 6.67 Hz, 2H, NCH₂CH₂(CH₂)₁₅CH₃), 4.24 (t, J = 7.15 Hz, 2H, N-CH₂CH₂(CH₂)₁₅CH₃), 6.65 (s br, 1H, H-8), 7.31 (m, 3H, H-3′, H-4′ e H-5′), 7.33 (d, J = 3.00 Hz, 1H, H-2), 7.65 (d, J = 7.29 Hz, 1H, H-4), 7.67 (m, 2H, H-2′ e H-6′), 7.68 (d, J = 9.00 Hz, 1H, H-5), 7.84 (d, J = 2.91 Hz, 1H, H-1), ppm. ¹³C NMR (75 MHz, CDCl₃): δ 14.02 (N-CH₂CH₂(CH₂)₁₅CH₃), 22.51 (NCH₂CH₂(CH₂)₁₅CH₃), 26.77 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.07 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.17 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.18 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.33 (NCH₂CH₂(CH₂)₁₅CH₃), 29.34 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.38 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.43 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.46 (N-CH₂CH₂(CH₂)₁₅CH₃), 29.50 (N-CH₂CH₂(CH₂)₁₅CH₃), 30.60 (NCH₂CH₂(CH₂)₁₅CH₃), 31.73 (N-CH₂CH₂(CH₂)₁₅CH₃), 46.56 (N-CH₂CH₂(CH₂)₁₅CH₃), 104.42 (C-1), 108.28 (C-8), 112.66 (C-4), 115.48 (C-5),117.75 (C-9a),123.16 (C-9b), 127.10 (C-2′), 127.60 (C-6′), 128.31 (C-3′), 128.74 (C-5′), 129.92 (C-2), 131.66 (C-4′), 134.75 (C-1′), 136.97 (C-3a), 139.18 (C-5a), 148.33 (C-7), 176.28 (C-9) ppm; IR (KBr): ν = 3403.42 (NH), 3090 (aliphatic C-H), 2903.12 (aliphatic C-H), 1651.15 (C==O), 1513.68 (C==C) cm⁻¹; UV-Vis (H₂O): 225.3 nm (A = 200.81 mAU), 276.10 nm (A = 197.23 mAU), 353.05 nm (76.28 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 458 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₃₅H₄₉N₂O⁺, 513.3845; found, 513.3945; RP-C18 HPLC: t_R = 31.711 min, 98.02%.

4.1.5.3. 3-ethyl-7-(naphthalen-1-yl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (21)

Compound 21 was prepared as described for compound 19 by reacting 0.877 g (2.28 mmol) of the appropriate phenylacrylate derivative 15 to yield 0.150 g of a yellow powder. Yield: 19.4%; Rf: 0.66 (light blue fluorescent spot, CHCl₃/methanol, 9:1); mp = 219 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 1.41 (t, J = 7.20 Hz, 3H, NCH₂CH₃), 4.34 (q, J = 7.22 Hz, 2H, NCH₂CH₃), 6.20 (s br, 1H, H-8), 7.43 (dd, J = 8.81 Hz, 1H, H-5), 7.52–7.75 (m, 5H, H-3′, H-4′, H-5′, H-6′ and H-7′), 7.59 (m, 1H, H-1) 7.85 (m, 1H, H-2), 7.90 (dd, J = 8.95, 0.64 Hz, 1H, H-4), 8.08 (m, J = 8.56, 2.14 Hz, 1H, H-8′), 8.13 (m, J = 8.23 Hz, 1H, H-2′), 12.01 (s br, 1H, NH) ppm; ¹³C NMR (101 MHz, DMSO-d₆): d 16.39 (NCH₂H₃), 41.03 (NCH₂CH₃), 104.10 (C-1), 111.58 (C-8), 112.53 (C-5), 115.98 (C-4), 118.31 (C-9a), 123.64 (C-9b), 125.30 (C-2), 125.87 (aromatic-C), 126.94 (aromatic-C), 127.64 (aromatic-C), 127.77 (aromatic-C), 128.64 (aromatic-C), 128.92 (C-8′), 130.12 (C-2′), 131.06 (C-8′a), 131.46 (C-4′a), 132.44 (C-3a), 133.61 (C-1′), 133.81 (C-7), 147.37 (C-5a), 178.15 (CO) ppm; IR (KBr): ν = 3436.75 (NH), 3024 (aliphatic C-H), 2974.66 (aliphatic C-H), 1608.39 (C==O), 1511.10 (C==C) cm⁻¹; UV-Vis (H₂O): 292 nm (A = 899 mAU), 340 nm (A = 501 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 479 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₂₃H₁₉N₂O⁺, 339.1492; found, 339.1540; RP-C18 HPLC: t_R = 14.34 min, 95.13%.

4.1.5.4. 3-ethyl-7-(naphthalen-3-yl)-3H-pyrrolo[3,2-f]quinoline-9(6H)-one (22)

Compound 22 was prepared as described for compound 19 by reacting 0.297 g (0.77 mmol) of the appropriate phenylacrylate derivative 16 to yield 0.270 g of a crude product, which was purified by flash column chromatography (eluent CHCl₃/methanol, 95:5) to yield 0.081 g of a dark yellow powder. Yield: 31%; Rf: 0.43 (blue fluorescent spot, CHCl₃/methanol, 95:5); mp = 230 °C; ¹H NMR (400 MHz, CDCl₃) d 1.46 (t, J = 7.3 Hz, 3H, NCH₂CH₃), 4.19 (q, J = 7.3 Hz, 2H, NCH₂CH₃), 6.61 (s, 1H, H-8), 7.17 (m, 2H, H-1 and H-3′), 7.23 (d, J = 2.9 Hz, 1H, H-2), 7.44 (m, 5H, H-4, H-4′, H-5, H-5'and H-6′), 7.83 (d, J = 2.8 Hz, 1H, H-7′), 8.00–7.93 (m, 2H, H-8′ and H-1′), 11.24 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): d 15.90 (NCH₂CH₃), 41.21 (NCH₂CH₃), 104.82 (C-1), 108.46 (C-8), 113.13 (C-5), 115.46 (C-9a), 117.97 (C-4), 123.23 (C-9b), 124.18 (C-5′), 126.29 (C-6′), 126.72 (C-2), 127.15 (C-3′), 127.22 (C-4′), 127.39 (C-7′), 128.40 (C-8′), 128.44 (C-1′), 131.27 (C-8′a), 131.61 (C-4′a), 132.68 (C-2′), 133.40 (C-3a), 137.16 (C-5a), 148.56 (C-7), 178.27 (CO) ppm; IR (KBr): ν = 3432.76 (NH), 3034 (aliphatic C-H), 2955.07 (aliphatic C-H), 1605.36 (C==O), 1518.17 (C==C) cm⁻¹; UV-Vis (H₂O): 212 nm (A = 1.03 AU), 235.09 nm (A = 982.95 mAU), 269.99 nm (1.11 AU), 301.52 nm (A = 1 AU), 358.49 nm (A = 498.23 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 471.06 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₂₃H₁₉N₂O⁺, 339.1492; found, 339.1561; RP-C18 HPLC: t_R = 15.21 min, 95.3%.

4.1.5.5. 3-ethyl-7-(1,2,3,4-tetrahydronaphthalen-6-yl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (23)

Compound 23 was prepared as described for compound 19 by reacting 0.302 g (0.89 mmol) of the appropriate phenylacrylate derivative 17 to yield 0.095 g of a crude product, which was purified by flash column chromatography (eluent CHCl₃/methanol, 9:1) to yield 0.055 g of a brown powder. Yield: 18%; Rf: 0.65 (light blue fluorescent spot, CHCl₃/methanol, 9:1); mp = 228 °C; ¹H NMR (400 MHz, CDCl₃) δ 1.52–1.46 (m, 7H, NCH₂CH₃ and H-6′ e H-7′), 2.49–2.44 (m, 4H, H-5′ e H-8′), 4.24 (q, J = 7.3 Hz, 2H, NCH₂CH₃), 6.59 (s, 1H, H-8), 6.83 (d, J = 7.7 Hz, 1H, H-1), 7.37–7.23 (m, 3H, H-1′, H-3′ and H-2), 7.59 (d, J = 8.5 Hz, 1H, H-4), 7.82 (d, J = 2.9 Hz, 1H, H-4′), 7.96–7.87 (d, J = 8.5 Hz, 1H, H-5), 11.69 (s, 1H, NH) ppm. ¹³C NMR (101 MHz, CDCl₃): δ 28.47 (NCH₂CH₃), 33.94 (C-6′ and C-7′), 39.01 (C-5′ and C-8′), 48.74 (NCH₂CH₃), 101.98 (C-1), 106.26 (C-8) 107.99 (C-9a), 110.07 (C-4), 114.46 (C-5), 115.15 (C-9b), 117.65 (C-3′), 117.99 (C-1′), 119.32 (C-2), 120.77 (C-4′), 121.02 (C-8′a), 125.36 (C-3a), 125.82 (C-2′), 127.18 (C-4'a), 134.86 (C-5a), 158.58 (C-7), 176.24 (CO) ppm; IR (KBr): ν = 3434.28 (NH), 3089 (aliphatic C-H), 2925.98 (aliphatic C-H), 1608.75 (C==O), 1508.04 (C==C) cm⁻¹; UV-Vis (H₂O): 216.26 nm (A = 543.13 mAU), 292.38 nm (A = 660.66 mAU), 355.26 nm (252.26 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 484.02 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₂₃H₂₃N₂O⁺, 343.1805; found, 343.1892; RP-C18 HPLC: t_R = 16.21 min, 95.36%.

4.1.5.6. 7-(benzo[d][1,3]dioxol-5-yl)-3-ethyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (24)

Compound 24 was prepared as described for compound 19 by reacting 0.556 g (1.47 mmol) of the appropriate phenylacrylate derivative 18 to yield 0.125 g of a crude product, which was purified by flash column chromatography (eluent CHCl₃/methanol, 95:5) to yield 0.086 g of a brown powder. Yield: 17.6%; Rf: 0.42 (light blue fluorescent spot, CHCl₃/methanol, 95:5); mp = 252 °C; ¹H NMR (400 MHz, DMSO) δ 1.41 (t, J = 7.2 Hz, 3H, NCH₂CH₃), 4.32 (q, J = 7.2 Hz, 2H, NCH₂CH₃), 6.15 (s, 2H, OCH₂O), 6.42 (s, 1H, H-8), 7.12 (d, J = 8.1 Hz, 1H, H-1), 7.45–7.38 (m, 2H, H-4′ and H-2), 7.45 (d, J = 1.6 Hz, 1H, H-1′), 7.50 (m, 1H, H-4), 7.57 (d, J = 8.9 Hz, 1H, H-5), 7.88 (d, J = 9.0 Hz, 1H, H-3′), 11.27 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 20.69 (NCH₂CH₃), 40.67 (NCH₂CH₃), 95.18 (OCH₂O), 104.82 (C-1), 106.67 (C-8), 107.56 (C-4′), 108.64 (C-4), 111.72 (C-9a), 113.30 (C-5), 113.38 (C-9b), 126.11 (C-3′), 126.46 (C-1′), 126.47 (C-2), 127.91 (C-3a), 128.04 (C-2a), 128.10 (C-5a), 136.45 (C-4′b), 136.53 (C-4′a), 143.76 (C-7), 160.49 (CO) ppm; IR (KBr): ν = 3446.49 (NH), 2923.24 (aliphatic C-H), 1627.04 (C==O), 1490.04 (C==C, 1255.78 (C-O) cm⁻¹; UV-Vis (H₂O): 220.66 nm (A = 337.43 mAU), 293.52 nm (A = 305.54 mAU), 363.07 nm (206.86 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 471.96 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for ${\mathrm{C}}_{20}{\mathrm{H}}_{17}{\mathrm{N}}_2{\mathrm{I}}_3^{+}$, 333.1234; found, 333.1337; RP-C18 HPLC: t_R = 13.49 min, 98.01%.

4.1.5.7. 1-ethyl-7-phenyl-1H-pyrrolo[2,3-f]quinolin-9(6H)-one (26) and 1-ethyl-7-phenyl-1H- pyrrolo[3,2-g]quinolin-5(8H)-one (27)

Compounds 26 and 27 were prepared as described for compound 19 by reacting 0.290 g (0.87 mmol) of the appropriate phenylacrylate derivative 25 to yield 0.186 g of a crude product consisting of the two isomers 26 and 27. We were unable to separate the two isomers, which were characterised as a mixture. Yield: 18%; Rf: 0.47 (light blue fluorescent spot, CHCl₃/methanol, 95:5); mp = 214 °C; ¹H NMR (300 MHz, CDCl₃): δ 1.15 (t, J = 8.23 Hz, 3H), 1.25 (t, J = 8.23 Hz, 3H), 4.11 (q, J = 7.80 Hz, 2H), 4.12 (q, J = 7.81 Hz, 2H), 6.58 (s, 1H), 6.32 (s, 1H), 7.36 (s, 1H), 7.41 (t, J = 2.05 Hz, 1H), 7.61 (m, 2H), 7.66 (d, J = 8.66 Hz, 2H), 7.69 (m, J = 8.23 Hz, 2H), 7.72 (d, J = 3.03 Hz, 1H), 7.80 (m, J = 8.22 Hz, 2H), 7.92 (d, J = 9.46 Hz, 1H), 7.98 (m, 4H), 8.02 (d, J = 9.17 Hz, 1H), 8.14 (s, 1H), 11.67 (s, 1H, NH), 11.69 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 15.34, 15.93, 40.23, 40.34, 104.52, 105.27, 106.95, 112.24, 113.50, 118.56, 119.39, 119.65, 121.42, 121.55, 124.83, 126.29, 126.67, 126.88, 127.38, 127.59, 127.92,128.09,128.48,128.99,129.06,129.37,129.99,130.43,130.74, 131.60, 133.72, 134.02, 136.93, 137.25, 139.86, 150.67, 152.13, 179.12, 180.02 ppm. IR (KBr): ν = 3422.11 (NH), 3082.51 (aromatic C-H) 2927.42 (aliphatic C-H), 1539.89 (C==C) cm⁻¹; UV-Vis (H₂O): 219.54 nm (A = 207.13mAU), 284.85 nm (A = 271 mAU), 296.06 nm (596.01 mAU), 359.25 nm (168.32 mAU) 377.68 (323.25 mAU); fluorescence (MeOH): λ_exc = 350 nm, λ_ems = 473.93 nm; HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₁₉H₁₇N₂O⁺, 289.1341; found, 289.1440; RP-C18 HPLC: t_R1 = 13.49 min, 49.52%, t_R2 = 19.23 min, 50.48%.

4.1.6. 1-ethyl-8-phenyl-1H-pyrrolo[3,2-h]quinolin-6(9H)-one (29) and (Z)-2-phenyl-[1,4]diazepino[3,2,1-hi]indol-4(1H)-one (30)

Compounds 29 and 30 were prepared as described for compound 19 by reacting 1.261 g (3.77 mmol) of the appropriate phenylacrylate derivative 28 to yield 0.180 g of a crude product consisting of the two isomers 29 and 30. The two isomers were separated by flash-column chromatography (eluent CHCl₃/methanol, 95:5) to yield 11 mg of compound 29 and 0.130 g of compound 30. Compound 29: Rf: 0.64 (light blue fluorescent spot, CHCl₃/methanol, 95:5); HRMS (ESI-MS, 140 eV): m/z [M+H]⁺ calculated for C₁₉H₁₇N₂O⁺, 289.1335; found, 289.1437; ¹H NMR (300 MHz, DMSO-d₆): δ 1.39 (t, J = 7.12 Hz, 3H, NCH₂CH₃), 4.27 (q, J = 7.11 Hz, 2H, NCH₂CH₃), 6.45 (s, 1H); 7.42 (d, J = 3.81 Hz, 1H), 7.47 (d, J = 3.21 Hz, 1H), 7.57 (d, 1H, J = 8.96 Hz), 7.65 (m, 3H), 7.89 (m, 2H), 7.97 (d, J = 8.96 Hz, 1H), 11.83 (s, 1H) ppm; ¹³C NMR (75 MHz, DMSO-d₆): 33.24, 49.95, 101.20, 108.02, 108.74, 117.39, 117.82, 128.39, 128.98, 139.14, 139.95, 141.92, 147.02, 150.31 ppm; Compound 30: Yield: 13.3%; Rf: 0.79 (CHCl₃/methanol, 95:5); ¹H NMR (300 MHz, DMSO-d₆): δ 6.75 (s, 1H, H-3), 7.13 (m, 3H, 3′-, 4′- and 5′-H), 7.25 (d, J = 2.73 Hz, 1H, H-6), 7.50 (d, J = 2.73 Hz, 1H, H-5), 7.75 (d, 1H, J = 9.00 Hz, 1H, H-7), 7.85 (m, 1H, H-8), 8.10 (d, J = 9.00 Hz, 1H, H-9), 8.33 (m, 2H, 2′- and 6′-H), 11.81 (s, 1H, NH) ppm; ¹³C NMR (75 MHz, DMSO-d₆): δ 98.03 (C-3),102.12 (C-6),125.87 (C-5),128.58 (C-4′), 128.85 (C-2′ e C-6′), 129.30 (C-3′ e C-5′), 130.51 (C-6a), 131.74 (C-7), 133.28 (C-8), 133.77 (C-9), 137.25 (C-1′), 160.73 (C-9a), 161.31 (C-2), 170.38 (C-4), 192.87 (C-9b) ppm; IR (KBr): ν = 3447.85 (NH), 3098.88 (aromatic C-H), 1740.47 (C=ONR1R2 stretching), 1626.09 (C=ONR1R2 stretching), 1548.79 cm-1 (C==C); UV-Vis (H₂O): 222.36 nm (A = 635.15 mAU), 357.76 nm (A = 894.41 mAU); ESI-MS: m/z [M+H]⁺ calculated for C₁₇H₁₃N₂O⁺, 261.1022, found 293.0904 (M = H = CH₃OH]⁺; RP-C18 HPLC: t_R1 = 18.03 min, 95.01%.

4.2. Biological assays

4.2.1. Cell growth conditions and antiproliferative assay

Human T-cell leukemia (Jurkat and CEM), human B-cell leukemia (RS4; 11) and human myeloid leukemia (Kasumi-1) cells were grown in RPMI-1640 medium (Gibco, Milano, Italy). Breast adenocarcinoma (MDA-MB-231), human cervix carcinoma (HeLa), non-small cell lung adenocarcinoma (A549) and human colon adenocarcinoma (HT-29) cells were grown in DMEM medium (Gibco, Milano, Italy), all supplemented with 115 units/mL penicillin G (Gibco, Milano, Italy), 115 µg/mL streptomycin (Invitrogen, Milano, Italy), and 10% fetal bovine serum (Invitrogen, Milano, Italy). Stock solutions (10 mM) of the different compounds were obtained by dissolving them in DMSO. Individual wells of a 96-well tissue culture microtiter plate were inoculated with 100 µL of complete medium containing 8 × 10³ cells. The plates were incubated at 37 °C in a humidified 5% CO₂ incubator for 18 h prior to the experiments. After medium removal, a 100 µL aliquot of fresh medium containing the test compound at a varying concentration was added to each well in triplicate and incubated at 37 °C for 72 h. Cell viability was assayed by the (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test as previously described [22]. The GI₅₀ was defined as the compound concentration required to inhibit cell proliferation by 50%.

CEM^Vbl−100 cells are a multidrug-resistant line selected against vinblastine and were a kind gift of Dr. G. Arancia (Istituto Superiore di Sanità, Rome, Italy). They were grown in RPMI-1640 medium supplemented with 100 ng/mL of vinblastine.

Peripheral blood lymphocytes (PBL) from healthy donors were obtained by separation on a Lymphoprep (Fresenius KABI Norge AS) gradient. After extensive washing, cells were resuspended (1.0 × 10⁶ cells/mL) in RPMI-1640 with 10% fetal bovine serum and incubated overnight. For cytotoxicity evaluations in proliferating PBL cultures, non-adherent cells were resuspended at 5 × 10⁵ cells/mL in growth medium containing 2.5 µg/mL PHA (Irvine Scientific). Different concentrations of the test compounds were added, and viability was determined 72 h later by the MTT test. For cytotoxicity evaluations in resting PBL cultures, non-adherent cells were resuspended (5 × 10⁵ cells/mL) and treated for 72 h with the test compounds, as described above.

4.2.2. Effects on tubulin polymerization and on colchicine binding to tubulin

To evaluate the effect of the compounds on tubulin assembly in vitro [12], varying concentrations of compounds were pre-incubated with 10 µM bovine brain tubulin in 0.8 M monosodium glutamate (from a 2 M stock solution adjusted to pH 6.6 with HCl) for 15 min at 30 °C and then cooled to 0 °C. After addition of 0.4mM GTP (final concentration), the mixtures were transferred to 0 °C cuvettes in a recording spectrophotometer equipped with an electronic temperature controller and warmed to 30 °C. Tubulin assembly was followed turbidimetrically at 350 nm. The IC₅₀ was defined as the compound concentration that inhibited the extent of assembly by 50% after a 20 min incubation. The ability of the test compounds to inhibit colchicine binding to tubulin was measured as described [13], with the reaction mixtures containing 1 µM tubulin, 5 µM [³H]colchicine and 1 or 5 µM test compound.

4.2.3. Molecular modeling

Compounds in Table 1 were built and their partial charges calculated after semi-empirical (PM6) energy minimization using MOE2016 [32, 33]. The molecular docking studies were based on the protocol recently reported for this target [8]. Briefly, the more suitable docking protocolwas identified using a benchmark over 14 protein/scoring protocol using DockBench 1.01 [34], a tool that compared the performance of 14 different posing/scoring protocols. Each ligand was docked 20 times using GOLD using PLP [35], the virtual screening tool of DockBench adopting the parameters already used in the benchmark study. The SARs of compounds 26–27 were not investigated by molecular docking because biological data was not available for these compounds.

To facilitate the visualization and analysis of data obtained from the docking simulations, we produced a video that shows the most relevant docking data, such as docking poses, per residue IEhyd and IEele data, experimental binding data and scoring values. Details for the video production were recently reported [9]. The logP values were predicted by the Stardrop software package [36].

4.2.4. Flow cytometric analysis of cell cycle distribution

5 × 10⁵ HeLa cells were treated with different concentrations of the test compounds for 24 h. After the incubation period, the cells were collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were then treated with lysis buffer containing RNase A and 0.1% Triton X-100 and stained with PI. Samples were analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems).

4.2.5. Apoptosis assay

Cell death was determined by flow cytometry of cells double stained with annexin V/FITC and PI. The Coulter Cytomics FC500 (Beckman Coulter) was used to measure the surface exposure of PS on apoptotic cells according to the manufacturer's instructions (Annexin-V Fluos, Roche Diagnostics).

4.2.6. Analysis of mitochondrial potential and ROS

The mitochondrial membrane potential was measured with the lipophilic cation JC-1 (Molecular Probes, Eugene, OR, USA), while the production of ROS was followed by flow cytometry using the fluorescent dyes H₂DCFDA (Molecular Probes), as previously described [23].

4.2.7. Western blot analysis

HeLa cells were incubated in the presence of 23 and, after different times, were collected, centrifuged, and washed two times with ice cold phosphate buffered saline (PBS). The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 15000 × g at 4 °C for 10 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal amounts of protein (10 µg) were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Criterion Precast, BioRad, Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were blocked with a bovine serum albumin solution (5% in Tween PBS 1X), and the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against Bcl-2, PARP, cdc25c, cyclin B, p-cdc2^Tyr15, Mcl-1 (all from Cell Signaling) and γ-tubulin (Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with peroxidase labeled secondary antibodies for 60 min. All membranes were visualized using ECL Select (GE Healthcare), and images were acquired using an Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To ensure equal protein loading, each membrane was stripped and reprobed with anti-γ-tubulin antibody.